1. Field of the Invention
This invention relates generally to the field of biochemistry and particularly to heterobifunctional coupling agents for making a wide array of molecular conjugates having numerous applications. More specifically, the agents contain a sterically hindered thiol, linked through a spacer arm to a second group reactive toward nucleophiles such as 1.degree. and 2.degree. amines or reactive thiols present on biological and organic materials. The coupling agents are useful for making conjugates containing a sterically hindered disulfide linkage, which conjugates are especially valuable for certain in vivo applications, such as targeted delivery of immunotoxins, drugs and radionuclides for cancer therapy and diagnosis.
2. Background Art
Conjugates between molecules with significantly different chemical or biological activities find broad use in analytical chemistry, clinical chemistry, and medicine. Enzymes directly or indirectly linked to antibodies find common use in immunoassays [Ishikawa et al. (1983) Journal of Immunoassay 4, 209-327]. Enzymes directly or indirectly linked to nucleic acid probes find increasing use in nucleic acid hybridization assays [Sheldon et al. [1987) Clin. Chem. 33, 1368-1371]. Conjugates between different antibodies may become therapeutically useful in antibody-dependent cell-mediated cytotoxicity [Titus et al. [1987) J. Immunol. 138, 4018-4022], a potential method for treating cancer, auto-immune disease, and immunological rejection reactions following tissue transplantation. The same therapeutic applications are envisioned for conjugates between toxins and antibodies, known as immunotoxins [Vitetta et al. (1987) Science 238, 1098-1104], as well as conjugates between antibodies and other therapeutic agents, including radionuclides and drugs of relatively low molecular weight. However, the total field of application of molecular conjugates is limited only by the imagination, as there are so many molecular functions, and within the functional domain of binding reactions, so many molecules with useful binding specificities (e.g., lectins for specific carbohydrates, hormones and cytokines for specific receptors, Staphylococcus protein A and certain complement components for immunoglobulins), that the total number of useful functional combinations is hard to count.
In some applications of molecular conjugates it is beneficial to use a crosslink between conjugated molecules which is cleavable under predictable or controlled conditions. In the field of pharmaceutical chemistry, it generally has been assumed that most effective immunotoxins require a cleavable bond between the toxin and the antibody which targets the toxin to a specific class of cells. Such immunotoxins are thought to operate by a multi-step pathway: binding to the cell surface, uptake into the interior of the cell, cleavage of the crosslink between antibody and toxin, and cell killing by the released toxin.
Three forms of chemical cleavability, generally applicable to molecular conjugation, have been engineered into immunotoxins. One uses an acid-labile crosslink, exploiting the fact that some of the intracellular compartments receiving internalized immunotoxins have pH values several pH units lower than that outside the cell. [Blatter et al. (1985) Biochemistry 24, 1517-1524]. A second cleavability tactic is to employ a peptide crosslinked with an amino acid sequence recognized by a specific protease [U.S. Pat. No. 4,571,958]. A third is the use of disulfide-containing crosslinks between antibody and toxin. The crosslinks may be cleaved rapidly upon addition of a relatively low (often approximately stoichiometric) concentration of a thiol. In as much as the thiol concentration in the extracellular fluid (e.g., blood plasma or lymph) is in the micromolar range, the intracellular thiol concentration exceeds 1 mM, largely due to the tripeptide, glutathione [Meister and Anderson (1983) Annual Review of Biochemistry 52, 711-760, see especially pp. 715-718]. Disulfide linked conjugates such as immunotoxins survive circulation in the blood well on the time scale of at least a few hours, yet are rapidly cleaved to release active toxin once they have been bound to and internalized by target cells.
U.S. Pat. No. 4,340,535 discloses such disulfide-crosslinked immunotoxins for the case in which the toxin is the ricin A chain and the antibody is either a whole immunoglobulin or an immunoglobulin fragment with binding specificity for an antigen carried by a cell. U.S. Pat. Nos. 4,350,626 and 4,450,154 claim immunotoxins in which a Fab [or Fab'] fragment of a tumor-specific antibody is coupled to the ricin A chain, with coupling occurring between cysteine-derived thiols on the two proteins, with or without an intervening bifunctional crosslinking group. U.S. Pat. Nos. 4,357,273 and 4,638,049 describe the analogous immunotoxins with diphtheria toxin being replaced by ricin A chain. U.S. Pat. No. 4,534,211 discloses conjugates where cytotoxic substances are attached to a cell-specific antibody or its fragment via at least one sulfur atom. The first of the above-named patents requires a disulfide bond within the crosslink. The last four allow such cleavable linkages to be made. None teaches how a sterically hindered disulfide bond might be made.
Recent pharmacokinetic studies of disulfide-linked immunotoxins show that they are less inert toward cleavage in the extracellular circulation than previously was thought [Blakey et al. (1987) Cancer Research 47, 947-952; Worrell et al. [1986) Anti-Cancer Drug Design 1, 179-188; Letvin et al. (1986) J. Clin. Invest. 77, 977-984]. Of the conjugate which has not been taken up from the blood by the various tissues, a significant fraction has been cleaved within eight hours of intravenous administration; essentially all has been split within 24 hours, long before the opportunity to kill target cells has been exhausted. This destructive side reaction may be the major limit to immunotoxin efficacy in vivo, because antibody cannot direct toxin to the target cells once the two molecules have separated. In addition, the antibody released from cleaved conjugate may compete with intact immunotoxin for cell surface binding sites. Accordingly, pharmaceutical science might be advanced greatly by the design of crosslinking agents with enhanced resistance to cleavage under extracellular conditions, which retain sufficient lability to break down on an effective time scale intracellularly.
Crosslinking of molecules, especially protein molecules, can be performed with homobifunctional or heterobifunctional reagents. The former require the molecules to be joined to have the same reactive groups. Because of this limitation homobifunctional reagents find little use in the modern art of macromolecular conjugation. Heterobifunctional crosslinking reagents require one of the molecules to be joined, hereafter called Partner B, to possess a reactive group not found on the other, hereafter called Partner A, or else require that one of the two functional groups be blocked or otherwise greatly reduced in reactivity while the other group is reacted with Partner A. In a typical two-step process for forming heteroconjugates, Partner A is reacted with the heterobifunctional reagent to form a derivatized Partner A molecule. If the unreacted functional group of the crosslinker is blocked, it is then deprotected. After deprotecting, Partner B is coupled to derivatized Partner A to form the conjugate. Primary amino groups on Partner A are reacted with an activated carboxylate or imidate group on the crosslinker in the derivatization step, and a reactive thiol or a blocked and activated thiol at the other end of the crosslinker is reacted with an electrophilic group or with a reactive thiol, respectively, on Partner B. When the crosslinker possesses a reactive thiol, the electrophile on Partner B preferably will be a blocked and activated thiol, a maleimide, or a halomethylene carbonyl (e.g., bromoacetyl or iodoacetyl) group. Because biological macromolecules do not naturally contain such electrophiles, they must be added to Partner B by a separate derivatization reaction. When the crosslinker possesses a blocked and activated thiol, the thiol on Partner B with which it reacts may be native to Partner B. Only when a thiol is reacted with a blocked and activated thiol can one be confident of forming a heteroconjugate with a cleavable disulfide linkage; which partner supplies which thiol species does not affect final conjugate structure.
Until recently, three heterobifunctional crosslinking agents were used almost to the complete exclusion of others in the preparation of disulfide-linked conjugates, including immunotoxins: N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP) [Carlson et al. (1978) Biochem. J. 173, 727-737], 2-iminothiolane (IT) [Jue et al. (1978) Biochemistry 17, 5399-5406], and S-acetyl mercaptosuccinic anhydride (SAMSA) [Klotz and Heiney (1962) Arch. Biochem. Biophys. 96, 605-612]. All three react preferentially with primary amines (e.g., lysine side chains) to form an amide or amidine group which links a thiol to the derivatized molecule (e.g., a protein, such as an antibody) via a connecting short spacer arm, one to three carbon atoms long. The differences among these molecules illustrate the tactical choices in making disulfide-linked conjugates.
Molecules derivatized with SPDP possess a blocked and activated thiol, ready for attack by a thiol on another molecule to generate a disulfide-linked conjugate between the two molecules. Alternatively, treatment with a sufficient concentration of a low-molecular weight thiol such as 2-mercaptoethanol or dithiothreitol displaces the blocking group to leave a reactive thiol on the derivatized molecule, which can attack a blocked and activated thiol on another molecule to create a conjugate identical in structure to the one just described, above. Either way, the sulfur atom which SPDP contributes to the final disulfide linkage experiences minimal steric hinderance, being attached to a methylene group of a straight-chain spacer arm. Molecules derivatized with IT possess a reactive thiol, which either can react with a blocked and activated thiol on another molecule or can be blocked and activated itself, by reaction with a chromogenic aryldisulfide, such as 2,2'-dithiodipyridine, 4,4'-dithiodipyridine, or 5,5'-dithio-bis(2-nitrobenzoic acid). Other reagents for blocking thiols which may create various degrees of activation toward further thiol-disulfide exchange include alkyl alkonethiolsulfonates, alkoxycarbonylalkyl disulfides, and various sulfenyl chlorides [Smith et al. (1975) Biochemistry 14, 766-771, Carlsson et al., supra]. Here too, the sulfur atom which the crosslinker contributes to the final conjugate is attached to a methylene group of a straight-chain spacer arm. Molecules derivatized with SAMSA possess a blocked thiol which is not activated toward reaction with another thiol. The blocking acyl group must be displaced with a strong nucleophile, most commonly hydroxylamine, to release a reactive thiol, which then can be reacted as in the case of IT. However, this thiol is sterically more hindered than in the case of SPDP or IT, as either a carboxylate or a carboxymethyl group branches from the spacer arm at the carbon atom to which the thiol is attached (a position "alpha" to the thiol). The ambiquity with regard to the branching group derives from the asymmetry of SAMSA, which permits the nucleophilic amine to react with either a carbonyl group adjacent to the sulfur-bearing carbon atom or a carbonyl group one carbon atom further away. In addition, the SAMSA-originated thiol may be chemically activated or deactivated by the negative charge of the carboxylate or carboxymethyl branch; the exact nature of this neighboring-group effect probably will depend on the specific reaction involving the thiol.
Recently two novel families of crosslinking agents have been disclosed which contain blocked thiols and are singly branched alpha to the thiol in the manner of SAMSA, and which have been used to couple molecules via sterically hindered disulfide bonds. N-succinimidyl 3-(2-pyridyldithio)butyrate (SPDB) [Worrell et al., supra] is identical in structure to SPDP except that it contain a single methyl-group branch alpha to the sulfur atom which is blocked and activated by 2-thiopyridine. SMPT and SMBT [Thorpe et al. (1987] Cancer Research 47, 5924-5931] contain a phenylmethyl spacer arm between an N-hydroxysuccinimide-activated carboxyl group and the blocked thiol; both the thiol and a single methyl-group branch are attached to the aliphatic carbon of the spacer arm. The SMBT thiol is blocked by sulfite, to form a thiosulfate, which must be cleaved to release a reactive thiol before crosslinking can occur. The SMPT thiol is blocked and activated via a disulfide bond to 2-thiopyridine, in the manner of SPDP. Data of Thorpe et al. [supra] suggest that the benzene ring in the SMBT and SMPT spacer arm hinders thiol reactivity more than do the aliphatic straight-chain spacers of SPDP, IT, or SPDB, presumably because it creates branching beta to the thiol and possibly because of reduced flexibility. To date, SPDB and SMPT usage has been reported only in coupling reactions where the reagent-derivatized antibody is reacted with another molecule bearing a free thiol. Deblocking of the reagents for thiol attack on the activated thiol of another molecule appears not to have been done.
The important functional advantage of these novel disulfide-creating crosslinkers singly branched at the alpha carbon atom is that they result in less easily cleaved disulfide bonds than do unbranched crosslinkers; comparison of SAMSA in this regard has not been reported. This result has been seen in model thiol-disulfide exchange reactions in vitro and studies of immunotoxin survival in ciruclation in vivo [Worrell et al., supra; Thorpe et al., supra]. The increased resistance to cleavage in vivo is correlated with significantly prolonged blood clearance times, which should enhance immunotoxin delivery to target cells, particularly if the latter are part of a solid tumor. However, neither Worrell et al. [supra] or Thorpe et al. [supra] have sucessfully synthesized immunotoxin with disulfide crosslinks singly branded alpha to the thiol, that show improved tumor growth-suppression or erodiation.
The development of sterically hindered disulfide crosslinks such as molecules with two methyl groups attached to the thiol-bearing carbon atom of the spacer arm has, before the instant invention, been unsuccessful. Worrell et al. [supra] prepared 3-(2-pyridyldithio]isovaleric acid, a potential intermediate in the synthesis of a doubly branched analogue of SPDP, but were unable to convert it into a crosslinker by activating the carboxyl group with N-hydroxysuccinimide.
Worrell et al. [supra] compared the reactivity toward thioldisulfide exchange of the sterically hindered activated disulfide in this molcule to the reactivities of analogues which were singly branched and unbranched alpha to the activated thiol. A single alpha methyl group reduced reactivity by one order of magnitude; double branching reduced reactivity by three orders of magnitude. If a way could be found to incorporate such a hindered disulfide into a conjugate, the latter might have radically improved survival in vivo over the disulfide-linked conjugates with single branching which represent the current state of the art. However, the possibility also exists that such conjugates would be so inert toward thiol-disulfide exchange that they could no longer effectively kill target cells.
Before the present invention, sterically hindered disulfide crosslinkers having two methyl groups attached to the thiol-bearing carbon atom of the spacer arm, have not been synthesized; and there was no guarantee that if such compounds could be made, they would result in immunotoxins with improved therapeutic properties. The present invention discloses methods for synthesizing sterically hindered disulfide crosslinkers that are distinctly unique from that which is known. Immunotoxin conjugates made with these coupling agents have improved survival in vivo and increased tumoricidal activity.